br Results br Discussion We examined

Results
Discussion We examined gene expression in human intestinal tissue across the developmental continuum in order to determine maturity of in-vitro-derived HIOs. Using global RNA expression data obtained by RNA-seq, several different analyses demonstrated that the HIO transcriptome closely resembles global gene expression in the human fetal intestine, strongly suggesting that HIOs more accurately represent fetal intestinal tissue than adult intestinal tissue. The adult intestine is a complex organ that performs many important metabolic and immunologic functions and hosts a tremendous population of indigenous bacteria. Our RNA-seq analysis supports the existing body of literature demonstrating that the human fetal intestine is immature with respect to advanced metabolic and host-defense functions, and that acquisition of these features is a hallmark of maturation to adult function. Similar to the human fetal gut, we have demonstrated that HIOs lack strong expression of many digestion-related genes and Paneth cells. We have further demonstrated that HIOs are competent to acquire these attributes of the adult intestine. This process requires transplantation into an in vivo context, suggesting that our in vitro conditions lack important cues for maturation. We suspect that cues from multiple sources are required to induce full maturation. For example, animal studies have highlighted the importance of intestinal microbial colonization in enhancing intestinal function, development of the intestinal immune system, and protection against pathogenic infection (Cash et al., 2006; Hooper, 2004; Hooper et al., 1999, 2001, 2012; Hudault et al., 2001). Moreover, it was recently demonstrated that HIOs transplanted into the kidney PCI-34051 could respond to circulating factors (Watson et al., 2014). Our finding that OLFM4 is not highly expressed in human or mouse fetal intestine but marks ISCs in the adult intestine is intriguing and suggests that there may be significant differences between fetal and adult ISCs. Importantly, the two predominant pathways that regulate adult ISCs, Wnt and Notch, are both thought to be important in late fetal development in mice (Korinek et al., 1998; Tsai et al., 2014; VanDussen et al., 2012; Zhong et al., 2012). Thus, it is possible that acquisition of an adult ISC state occurs gradually across the developmental continuum, such that there are differences in ISC regulation in early fetal development, late fetal development, postnatally, and in adult life. At this time, careful studies across different developmental ages have not been carried out. It is also currently unclear if the gene expression differences between fetal and adult life are functionally significant because, for example, Olfm4 knockout animals do not show a phenotype (Schuijers et al., 2014). In addition to HIOs, other groups have established primary cell culture models of the human and mouse fetal intestine, called fetal enterospheres (Fordham et al., 2013; Mustata et al., 2013). Mouse fetal enterospheres only retain fetal properties for a limited time because they continue to develop into adult-like organoids. Therefore, it appears as though the developmental program may be hardwired into the epithelium that is removed from the fetal gut, and may pose limitations to the use of primary fetal tissue. While fetal enterospheres are an invaluable tool, our results show that HIOs offer a complementary system that may be more accessible to a wider portion of the research community. Given the difficulty or inability to obtain human fetal tissue for many researchers, in addition to the ethical and legal concerns of using this tissue in biomedical research, our data suggest that HIOs provide an appropriate alternative model.
Experimental Procedures
Author Contributions
Acknowledgments